Luciferase should be 1653 bp (its 3426 bp backbone should be discarded)

Perform ligation

S and X are compatible so we get Pend---backbone--E--X--promoter--RBS--SXscar--luciferase---S--Pend

The two Pends will also ligate, producing a circular desired plasmid

Transform competent TOP10 cells with ligation product

What happened

In the morning we were a bit disconsolate because there was nothing visible on the plates containing phK724 and phK555, and our transformation tests for the variant hns strains were less successful than we had hoped.

However our mood changed when Jim A told us we could not hope for many transformants with 20ul of strains that were not TOP10. We decided to retest the strains with 100 ul plated out.{{

AND we checked the 30 C incubator again and saw 2 colonies on one of the pHK555 plates, viewed under the camera they were clearly glowing so we gathered in the cold room and passed them around, as our eyes adapted we saw the colonies. We even saw a third glowing colony which we had not spotted on the plate.

Friday

Ligation appeared to have failed.
Jim A suggests that we heat to 65 degrees to melt short DNAs after restriction digest, plus that we clean up the DNA with a spin column without gel.